This present invention provides cDNA sequences and polypeptides having the enzyme CDP-diacylglycerol synthase (CDS) activity. CDS is also known as CTP:phosphatidate cytidyltransferase (EC2.7.7.41). The present invention further provides for isolation and production of polypeptides involved in phosphatidic acid metabolism and signaling in mammalian cells, in particular, the production of purified forms of CDS.
CDP-diacylglycerol (DAG) is an important branch point intermediate just downstream of phosphatidic acid (PA) in the pathways for biosynthesis of glycerophosphate-based phospholipids (Kent, Anal. Rev. Biocheni. 64: 315-343, 1995). In eukaryotic cells, PA, the precursor molecule for all glycerophospholipid, is converted either to CDP-DAG by CDP-DAG synthase (CDS) or to DAG by a phosphohydrolase. In mammalian cells, CDP-DAG is the precursor to phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL). Diacylglycerol is the precursor to triacylglycerol, phosphatidylethanolamine, and phosphatidylcholine in eukaryotic cells. Therefore, the partitioning of phosphatidic acid between CDP-diacylglycerol and diacylglycerol must be an important regulatory point in eukaryotic phospholipid metabolism (Shen et al., J. Biol. Chem. 271:789-795, 1996). In eukaryotic cells, CDP-diacylglycerol is required in the mitochondria for phosphatidylglycerol and cardiolipin synthesis and in the endoplasmic reticulum and possibly other organelles for the synthesis of phosphatidylinositol (PI). PI, in turn, is the precursor for the synthesis of a series of lipid second messengers, such as phosphatidylinositol-4,5-bisphosphate (PIP2), DAG and inositol-1,4,5-trisphosphate (IP3). Specifically, PIP2 is the substrate for phospholipase C that is activated in response to a wide variety of extracellular stimuli, leading to the generation of two lipid second messengers; namely, DAG for the activation of protein kinase C and 1P3 for the release of Ca++ from internal stores (Dowhan, Anal. Rev. Biochem. 66: 199-232, 1997).
The genes coding for CDS have been identified in E. coli (Icho et al, J. Biol. Chem. 260: 12078-12083, 1985), in yeast (Shen et al., J. Biol. Chem. 271:789-795, 1996), and in Drosophila (Wu et al., Nature 373:216-222, 1995). A human cDNA coding for CDS (hCDS1) is described by us herein and has been reported in Weeks et al., DNA Cell Biol. 16: 281-289, 1997. Moreover, Heacock et al., J. Neurochem. 67: 2200-2203, 1997 report cloning of a CDS1 from a human neuronal cell line. Furthermore, Lykidis et al., J. Biol. Chem 272:33402-33409 ,1997 and Halford et al., Genomics 54:140-144, 1998 both report DNA sequences suspected to encode a human cds2 protein, but these references fail to disclose either biological activity or an intact N-terminal region for the putative proteins.
It is of interest to isolate polynucleotides coding for human CDS and express them in mammalian cells to determine the potential roles of this enzyme in cellular function and use this enzyme as a target for the development of specific compounds that are modulators of its activity. With the advance in the understanding of disease processes, it has been found that many diseases result from the malfunction of intracellular signaling. This recognition has led to research and development of therapies based on the interception of signaling pathways in diseases (Levitzki, Curr. Opin. Cell Biol. 8:239-244, 1996). Compounds that modulate CDS activity, and hence modulate generation of a variety of lipid second messengers and signals involved in cell activation, are therefore of therapeutic interest generally, and of particular interest in the areas of inflammation and oncology:
The present invention provides cDNA sequences, polypeptide sequences, and transformed cells for producing isolated recombinant mammalian CDS. The present invention provides two novel human polypeptides and fragment thereof, having CDS activity. The polypeptides discovered herein are novel and will be called hCDS1 (human CDS1) and hCDS2 (human CDS2). CDS catalyzes the conversion of phosphatidic acid (PA) to CDP-diacylglycerol (CDP-DAG), which in turn is the precursor to phosphatidylinositol (PI), phosphatidylglycerol (PG) and cardiolipin (CL).
The present invention further provides nucleic acid sequences coding for expression of the novel CDS polypeptides and active fragments thereof. The invention further provides purified CDS mRNAs and antisense oligonucleotides for modulation of expression of the genes coding for CDS polypeptides. Assays for screening test compounds for their ability to modulate CDS activity are also provided.
Recombinant CDS is useful for screening candidate drug compounds that modulate CDS activity, particularly those compounds that activate or inhibit CDS activity. The present invention provides cDNA sequences encoding a polypeptide having CDS activity and comprising the DNA sequence set forth in SEQ ID NO. 1 (hCDS1), the DNA sequence (SEQ ID NO:11) set forth in FIG. 8 (hCDS2), shortened fragments thereof, or additional cDNA sequences which due to the degeneracy of the genetic code encode a polypeptide of SEQ ID NO. 2 (hCDS1), a polypeptide of FIG. 8 (hCDS2), or biologically active fragments thereof, or a sequence hybridizing thereto under high stringency conditions. The present invention further provides a polypeptide having CDS activity and comprising the amino acid sequence of SEQ ID NO. 2 (hCDS1), the amino acid sequence (SEQ ID NO:12) of FIG. 8 (hCDS2), or biologically active fragments thereof
Also provided by the present invention are vectors containing a DNA sequence encoding a mammalian CDS enzyme in operative association with an expression control sequence. Host cells, transformed with such vectors for use in producing recombinant CDS are also provided with the present invention. The inventive vectors and transformed cells are employed in a process for producing recombinant mammalian CDS. In this process, a cell line transformed with a cDNA sequence encoding a CDS enzyme in operative association with an expression control sequence, is cultured. The claimed process may employ a number of known cells as host cells for expression of the CDS polypeptide, including, for example, mammalian cells, yeast cells, insect cells and bacterial cells.
Another aspect of this invention provides a method for identifying a pharmaceutically-active compound by determining if a selected compound modulates the activity of CDS for converting PA to CDP-DAG. A compound having such activity is capable of modulating signaling kinase pathways and being a pharmaceutical compound useful for augmenting trilineage hematopoiesis after cytoreductive therapy and for anti-inflammatory activity in inhibiting the inflammatory cascade following hypoxia and reoxygenation injury (e.g., sepsis, trauma, ARDS, etc.).
The present invention further provides a transformed cell that expresses active mammalian CDS and further comprises a means for determining if a drug candidate compound is therapeutically active by modulating recombinant CDS activity.